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Histamine Release Induced by Dynorphin-(1-13) from Rat Mast Cells
Japan. J. Pharmacol. 35, 247-252
Histamine
247
(1984)
Release Induced by Dynorphin-(1-13) from Rat Mast Cells
KatsumiSUGIYAMA and HiroakiFURUTA Department of Pharmacology, Okayama University DentalSchool, 2-5-1Shikata-cho, Okayama 700,Japan Accepted March26,1984 Abstract-Dynorphin (Dyn) mast
is
induced cells in
a
histamine a dose-dependent
potent release
Dynorphin-(1-13)-induced and
the
release
Calcium 10-3
M
about
M)
enhanced release.
by that mast
Disodium Dyn,
cells.
Its
the
and
of
completed the
the
action
from range
of
although for
that
was
histamine
These
by
of
Dyn
the
not.
mediated
to
37•‹C,
than
by
inhibited did
appeared
at
concentration release
M)
not
sec
rat M.
dehydrogenase.
higher
maximum
was
isolated 10-7-10-5
10
lactate
leucine-enkephalin release
dynorphin-(1-13)
within
leakage
release, pH
synthetic
(5•~10-6-10-4
histamine mode
the
by
The
naloxone
Dyn-induced
A
by degranulation concentration
release
cromoglycate
but
peptide.
accompanied
the
indicate the
not
(10-5-10-3
release
of
histamine was
suppressed
7.3.
opioid
accompanied manner over
results
opioid
receptors
anaphylactic
histamine
release.
Dynorphin is a heptadecapeptide which contains a leucine-enkephalin sequence as its amino terminal end (1, 2). lmmunoreactive dynorphin has been shown to be distributed in the central nervous system, the pituitary gland and peripheral nervous system (3-5). A synthetic dynorphin-(1-13) (Dyn) is potent as natural dynorphin-(1-17) in bioassay (6), and it has been shown to produce analgesia and catalepsy (7), alteration in centrally stimulated gastric acid secretion (8) and spontaneous feeding (9). However, the physiological role of dynorphin is at present uncertain. In this study, we report that Dyn stimulates the isolated rat mast cells to extrude granules and release histamine by a exocytotic mechanism. A preliminary report of these data has been published (10). Materials and Methods 1. Mast cells and histamine release: Mast cells were collected from the peritoneal cavities of male Wistar rats (300-350g) and purified as previously described (11). The mast cells were suspended in a phosphatebuffered saline (PBS) consisting of 154 mM
NaCl,
2.7
mM
KCl.
0.9
KH2PO4-Na2HPO4, bovine
serum
cells
used For
1.5
ml
was
of
peptides was
ice-cold
The
at
histamine
the
of
was total
37•‹C,
and
was
after
a
chilled
at
PBS.
ice
bath
min
at
and 4•‹C.
Each
To
phological observed
changes under
contrast
microscope.
at
4•‹C.
kinetics
suspension
of
Dyn,
the
time
to of
release
the
at
25•‹C
or
reaction
intervals
volume
by of
transferred .3000•~g the
of
according Release
cell
was
was
and
min
at
an
min
5
higher
determine
buffer
37•‹C, adding
a percent
different
centrifuged
phosphate
at
mixture
15
study
the
tube
were
by
(12).
as
times
(105
PBS
reaction
To
addition
10
mast
supernatants
for
terminated
adding
the
release,
preincubated
min
for
content.
histamine
were
10
The
3000•~g
expressed
cell
of
for
was determined of Shore et al.
histamine of
the
cells
ml
terminated
in
precipitates the method
0.5
PBS.
centrifuged
of
mast
in
with
mM 0.01%
90%.
release,
reaction
6.7
and
purity
than
suspended
the
CaCl2,
7.2,
The
more
histamine
incubated and
albumin.
was
cells/ml)
mM
pH
the to
for
effect
of
an 15 pH,
used.
The
mor-
mast invert-type
cells
were phase-
248
K. Sugiyama
& H. Furuta
2. Lactate dehydrogenase-release assay: Lactate dehydrogenase (LDH) activity was determined according to the fluorimetric method, as previously described by Roy et al. (13). The LDH release was assessed in relation to fluorescence intensity obtained after addition of 0.01% Triton X-100. 3. Chemicals: Dynorphin-(1-13) and leucine-enkephalin was obtained from the Protein Research Foundation (Osaka, Japan): disodium cromoglycate from Fujisawa Pharmaceutical Co. (Osaka, Japan); naloxone hydrochloride from Sankyo Company (Tokyo, Japan). Other chemicals of reagent grade were obtained from standard commercial sources.
(mean•}S.E.)
Fig.
2.
(B)
after
Phase-contrast treatment
micrographs with
5•~10-6
of M for
mast
cells
5 min. •~500.
The
(10-5
M)
release
of
2.
Results 1. Induction of histamine release by Dyn: Synthetic Dyn induced histamine release from isolated mast cells in a dose-dependent manner over the concentration range 10-710-5 M (Fig. 1). Phase contrast observations of mast cells responding to Dyn showed granule extrusion (Fig. 2). Lactate dehydrogenase did not release in the response to Dyn. Spontaneous release of histamine and LDH from mast cells was 4.2+0.8%. n=4
and
tively.
Fig.
1.
rat
Mast with
three
separate
cells
Dyn.
to
histamine
cells were
Values
while
the
release:
for
induced
by
incubated represent
Dyn
n=4.
relationships mast
respecby
n=4,
3.2•}0.4%, of
n=4, induced
70.2•}5.3, was
Dose-response
37•‹C
response
release
course
from
(1-13).
in
was LDH
Time
release
2.3•}0.5%,
histamine
for
The
histamine dynorphin10
the
mean•}S.E.
(A)
before
mm
of
experiments.
dynorphin-(1-13).
at
and
Histamine response
to
onset
Dyn
at
release
was
25•‹C,
min
(Fig. 3.
3). Effect
of
release
by
Dyn
(Fig. atures
4), the below
release
than
4.
Effect
7.5
and
(Fig.
pH's
added
addition
At temperhistamine
simple
omission
of
the
cells
ions release with 10-5
and
calcium
from
reduced this
various
was
of
EDTA
release times
Ca2+-free
the
release
histamine
at
the M,
M
addition
the
in
or
Ca2+-concen-
of
However,
Dyn
6.5
5•~10-4
medium
by
to of
was
than
increased from
at
shows
histarelease
Calcium
increase
influenced
Ca2+
Histamine
histamine
further
response.
7
the
calcium:
cell-suspending
(10-3 M). Figure
by
after
medium.
the
Fig.
4.
by with 37•‹C
3.
Time
course
of
dynorphin-(1-13). Dyn (•œ).
experiments.
histamine Mast
(5•~10-6
M)
Values
represent
for
release
cells 10 the
were
min
at
meari•}S.E.
of
induced
were
temperature by
preincubated
and
further
Dyn
(5•~10-6
at
various
incubation
represent
M) the
on
the
histamine
dynorphin-(1-13).
or
mean•}S.E.
cells
for
5 min,
temperatures
was (•œ)
Mast
made
for
without of
10
Dyn
three
min
(•›).
with Values
experiments.
When
induced
(•›) of
Effect by
incubated or
three
5.
induced
incubated 25•‹C
Effect
release
Fig. Fig.
exposure of reached a time, apbasal level
5).
maximum
not
a
CaCl2 was added after a 10-sec cells to Dyn, histamine release maximum and decayed with proaching asymptotically the
temperature
lower
effect of Ca2+ concentration
on
releasing
pH:
37•‹C. 40•‹C,
Dyn-induced
The of
The
the
at
249
by Dynorphin
peptide
Dyn-induced
7.3,
of
reaching
tration.
while
the
on
being above
at
the
declined
of and
for
was
enhanced (Fig. 6). increase
sec,
dependent
suppressed
higher
in
approached
addition
optimum
release
largely
10
temperature was
rapid histamine
release
after
Induced
suppressed.
pH
mine
within
optimum 15•‹C and
was
The
was
maximal
histamine
1
M)
and
reached
the
plateau
(5•~10-6
37•‹C
Release
(•œ)
or
with of
for
10
without
various three
of
pH
on
the
histamine
dynorphin-(1-13). min Dyn pH's.
experiments.
at (•›) Values
Mast 37•‹C in
with a
Dyn
suspending
represent
release cells
the
were
(5•~10-6 medium mean•}S.E.
M)
250
K. Sugiyama
& H. Furuta
obtained in the absence of calcium. 5. Effect of inhibitors: Cromoglycate inhibited histamine release stimulated by Dyn in a dose-dependent manner (Fig. 8a). This inhibition was dependent upon the incubation time with cromoglycate before addition of Dyn (Fig. 8b). The addition of cromoglycate 30 sec before Dyn inhibited
markedly histamine release from mast cells, but such inhibition was abolished after a 2-min incubation with cromoglycate. Naloxone (10-6-10-4 M) and leucineenkephalin (10-7-10-4 M) had little effect
Fig.
7.
Time
release Fig.
6.
Effect
induced
by
incubated in
a
of
at
on
the
histamine
dynorphin-(1-13). 37•‹C
medium
trations.
calcium
for
Mast
10
containing
Values
min
with
CaCl2
represent
the
cells
Dyn in
release
M)
concen-
mean•}S.E.
of
Fig.
8.
different
37•‹C
in
(•›).
CaCl2
periods
three
(•œ).
experiments.
was
of
calcium
decay
added
dynorphin-(1-13).
were
(5•~10-6
varying
course
by
Mast
a
Ca2+-free
time
Values
cells
medium
(5•~10-4
of
was
exposure
represent
the
the
of
incubated
Dyn added
to
histamine
stimulation
were with
M)
after
of after
at
(5•~10-6 at
M)
different
Dyn
(5•~10-6
mean•}S.E.
of
M) three
experiments.
Inhibition
added
to times
by mast after
cromoglycate cells exposure
30
of sec to
histamine
before cromoglycate.
addition
release of Values
induced
Dyn
by
(5•~10-6
represent
dynorphin-(1-13) M) .
the
b:
mean•}S.E.
Dyn
. (5•~10-6 of three
a: M)
Cromoglycate was
added
experiments
.
at
Histamine
Release
on the mast cells and on the Dyn-induced histamine release. Discussion We have shown here that Dyn induces histamine release from isolated rat peritoneal mast cells concomitantly with their degranulation. This histamine release is not accompanied by leakage of LDH. The response to Dyn is dependent on temperature and pH in the reaction medium. Calcium enhances the histamine release induced by Dyn. These data indicate that Dyn causes the release of histamine by an exocytotic mechanism. The histamine release induced by Dyn was augmented by the extracellular calcium, but a significant release of histamine persisted in the absence of calcium. Addition of EDTA in the absence of calcium did not abolish the histamine release by Dyn. However, we could not observe the restoration of histamine release by calcium after prolonged treatment of the cells with EDTA, as previously observed by Douglas and Ueda (14). The stimulant effect of Dyn would therefore be that it acts by utilizing extracellular calcium or by mobilizing cellular calcium which mediates the releasing response. Calcium-induced histamine release from the cells activated by Dyn in the Ca2+-free medium decayed with time and decreased on further exposure to Dyn. The time sequence of inactivation was similar to the process of desensitization to antigen described by Foreman and Garland (15). They suggested that mast cells limited the entry of calcium after stimulation by shutting-down Ca channels opened by the stimulus. A similar observation on inactivation of compound 48/ 80-treated cells has been shown by Cochrane et al. (16). However, the mechanism of Ca channel inactivation in mast cells is not yet understood. Cromoglycate has been shown to inhibit exocytosis from mast cells stimulated with a variety of agents such as antigen (17), compound 48/80 (18, 19), dextran (20) and neurotensin (21). The histamine release induced by Dyn was also inhibited by cromoglycate in a dose-dependent fashion. This inhibitory action was effective by the treatment with this agent for very short
Induced
by Dynorphin
251
periods of time. These conform to a familiar pattern observed with various other agents in mast cells (22). Cromoglycate might have a stabilizing effect on the mast cell membrane involved in exocytosis. However, the mechanism of action of cromoglycate in the inhibitory effect is at present uncertain. The actions of Dyn in bioassay systems such as guinea pig ileum and mouse was deferens are antagonized by naloxone (6), and it has been shown that Dyn acts as a κ-receptor
agonist(23).Dyn-induced
hista-
mine release was not inhibited by both naloxone and leucine-enkephalin. The histamine release from the mast cells was also not induced by leucine-enkephalin alone. These findings indicate that histamine release by Dyn is not probably mediated by opioid receptors of the mast cells. Certain basic biologically active peptides are known to acts as histamine releasers from mast cells (21, 24, 25). Dynorphin contains the leucine-enkephalin sequence at its Nterminus, followed by its C-terminus in which basic amino acids predominate. Dynorphin-(1-13) contains five basic amino acids in its peptide structure. Therefore, the releasing response to Dyn may reflect the stimulating effect of basic peptides observed by Johnson and Erdos (24). References 1 Goldstein, A., Fischli, W., Lowney, L.1., Hunkapiller, M. and Hood, L.: Pocine pituitary dynorphin: Complete amino acid sequence of the biologically active heptadecapeptide. Proc. Natl. Acad. Sci. U.S.A. 78, 7219-7223 (1981) 2 Tachibana, S., Araki, K., Ohya, S. and Yoshida, S.: Isolation and structure of dynorphin, an opioid peptide, from porcin duodenum. Nature 295, 339-340 (1982) 3 Goldstein, A. and Ghazarossian, V.E.: Immunoreactive dynorphin in pituitary and brain. Proc. Natl. Acad. Sci. U.S.A. 77, 6207-6210 (1980) 4 Hollt, V., Haarman, I., Bovermann, K., Jerlicz, M. and Herz, A.: Dynorphin-related immunoreactive peptides in rat brain and pituitary. Neurosci. Lett. 18, 149-153 (1980) 5 Watson, S.J., Akil, H., Gharzarossian, V.Z. and Goldstein, A.: Dynorphin immunocytochemical localization in brain and peripheral nervous system: Preliminary studies. Proc. Natl. Acad.
252
K. Sugiyama
Sci. U.S.A. 78, 1260-1263 (1981) 6 Goldstein, A., Tachibana, S., Lowney, L.I., Hunkapiller, M. and Hood, L.: Dynorphin- (1-13), an extraodinarilly potent opioid peptide. Proc. Natl. Acad. Sci. U.S.A. 76, 6666-6670 (1979) 7 Herman, B.H., Leslie, F. and Goldstein, A.: Behavioral effects and in vivo degradation of intraventriculary administered dynorphin- (1-13) and D-Ala2-dynorphin-(1-11) in rat. Life Sci. 27, 883-892 (1980) 8 Morley, J.E., Levine, A.S. and Silvis, S.E.: Endogenous opiates inhibit gastric acid secretion induced by central administration of thyrotropin releasing hormone (TRH). Life Sci, 29, 293-297 (1981) 9 Morley, J.E. and Levine, A.S.: Dynorphin- (113) induces spontaneous feeding in rats. Life Sci. 18, 1901-1903 (1981) 10 Sugiyama, K. and Furuta, H.: Histamine release induced by dynorphin-(1-13) from rat mast cells. Japan. J. Pharmacol. 33, Supp. 59P (1983) 11 Sugiyama, K.: Calcium-dependent histamine release with degranulation from isolated rat mast cells by adenosine 5'-triphosphate. Japan. J. Pharmacol. 21, 209-226 (1971) 12 Shore, P.A., Burkhalter, A. and Cohn, V.H., Jr.: A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther. 127, 182-186 (1959) 13 Roy, D., Moran, D.M., Bryant, V., Stevenson, R. and Stanworth, D.R.: Further studies on histamine release from rat mast cells in vitro induced by peptides. Biochem. J. 191, 233-237 (1980) 14 Douglas, W.W. and Ueda, Y.: Mast cell secretion (histamine release) induced by 48/80: Calcium dependent exocytosis inhibited strongly by cytochalasin only when glycolysis is ratelimiting. J. Physiol. (Lond.) 234, 97P-98P (1973) 15 Foreman, J.C. and Garland, L.G.: Desensitization in the process of histamine secretion induced by antigen and dextran. J. Physiol. (Lond.) 239, 381-391 (1974) 16 Cochrane, D.E., Distel, D.L., Lansman, J.B. and Paterson, B.M.: Stimulas-secretion coupling in
& H. Furuta
17
18
19
20
21
22
23
rat mast cells: Inactivation of extracellular calcium dependent secretion. J. Physiol. (Lond.) 323, 423-435 (1982) Garland, L.G.: Effect of cromoglycate on anaphylactic histamine release from rat peritoneal mast cells. Br. J. Pharmacol. 49, 128-130 (1973) Orr, T.S.C., Hall, D.E., Gwilliam, J.M. and Cox, J.S.G.: The effect of disodium cromoglycate on the release of histamine and degranulation of rat mast cells induced by compound 48/80. Life Sci. 10,805-809 (1971) Spataro, A.C. and Bosmann, H.B.: Mechanism of action of disodium cromoglycate on mast cells calcium ion influx after a histamine releasing stimulus. Biochem. Pharmacol. 25, 505-509 (1976) Garland, L.G. and Mongar, J.L.: Inhibition by cromoglycate of histamine release from rat peritoneal mast cells induced by mixtures of dextran, phosphatidylserine and calcium ions. Br. J. Pharmacol. 50, 137-143 (1974) Carraway, R., Cachrane, D.E., Lansman, J.B., Leeman, S.E., Paterson, B.M. and Welch, H.J.: Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels. J. Physiol. (Lond.) 323, 403414 (1982) White, J.R. and Pearce, L.: Effect of antiallergic compound an anaphylactic histamine secretion from rat peritoneal mast cells in the presence and absence of exogenous calcium. Immunology 46, 361-367 (1982) Oka, K., Negishi, K., Suda, M., Sawa, A., Fujino, M. and Wakimasu, M.: Evidence that dynorphin-(1-13) acts an agonist on opioid κ-receptors.
Eur.
J.
Pharmacol.
77,
137-141
(1982) 24 Johnson, A.R. and Erdos, E.G.: Release of histamine from mast cells by vasoactive peptides. Proc. Soc. Exp. Biol. Med. 142, 1252-1256 (1973) 25 Theoharides, T.C. and Douglas, W.W.: Somatostatin induces histamine secretion from rat peritoneal mast cells. Endocrinology 102, 16371640 (1978)
Histamine
247
(1984)
Release Induced by Dynorphin-(1-13) from Rat Mast Cells
KatsumiSUGIYAMA and HiroakiFURUTA Department of Pharmacology, Okayama University DentalSchool, 2-5-1Shikata-cho, Okayama 700,Japan Accepted March26,1984 Abstract-Dynorphin (Dyn) mast
is
induced cells in
a
histamine a dose-dependent
potent release
Dynorphin-(1-13)-induced and
the
release
Calcium 10-3
M
about
M)
enhanced release.
by that mast
Disodium Dyn,
cells.
Its
the
and
of
completed the
the
action
from range
of
although for
that
was
histamine
These
by
of
Dyn
the
not.
mediated
to
37•‹C,
than
by
inhibited did
appeared
at
concentration release
M)
not
sec
rat M.
dehydrogenase.
higher
maximum
was
isolated 10-7-10-5
10
lactate
leucine-enkephalin release
dynorphin-(1-13)
within
leakage
release, pH
synthetic
(5•~10-6-10-4
histamine mode
the
by
The
naloxone
Dyn-induced
A
by degranulation concentration
release
cromoglycate
but
peptide.
accompanied
the
indicate the
not
(10-5-10-3
release
of
histamine was
suppressed
7.3.
opioid
accompanied manner over
results
opioid
receptors
anaphylactic
histamine
release.
Dynorphin is a heptadecapeptide which contains a leucine-enkephalin sequence as its amino terminal end (1, 2). lmmunoreactive dynorphin has been shown to be distributed in the central nervous system, the pituitary gland and peripheral nervous system (3-5). A synthetic dynorphin-(1-13) (Dyn) is potent as natural dynorphin-(1-17) in bioassay (6), and it has been shown to produce analgesia and catalepsy (7), alteration in centrally stimulated gastric acid secretion (8) and spontaneous feeding (9). However, the physiological role of dynorphin is at present uncertain. In this study, we report that Dyn stimulates the isolated rat mast cells to extrude granules and release histamine by a exocytotic mechanism. A preliminary report of these data has been published (10). Materials and Methods 1. Mast cells and histamine release: Mast cells were collected from the peritoneal cavities of male Wistar rats (300-350g) and purified as previously described (11). The mast cells were suspended in a phosphatebuffered saline (PBS) consisting of 154 mM
NaCl,
2.7
mM
KCl.
0.9
KH2PO4-Na2HPO4, bovine
serum
cells
used For
1.5
ml
was
of
peptides was
ice-cold
The
at
histamine
the
of
was total
37•‹C,
and
was
after
a
chilled
at
PBS.
ice
bath
min
at
and 4•‹C.
Each
To
phological observed
changes under
contrast
microscope.
at
4•‹C.
kinetics
suspension
of
Dyn,
the
time
to of
release
the
at
25•‹C
or
reaction
intervals
volume
by of
transferred .3000•~g the
of
according Release
cell
was
was
and
min
at
an
min
5
higher
determine
buffer
37•‹C, adding
a percent
different
centrifuged
phosphate
at
mixture
15
study
the
tube
were
by
(12).
as
times
(105
PBS
reaction
To
addition
10
mast
supernatants
for
terminated
adding
the
release,
preincubated
min
for
content.
histamine
were
10
The
3000•~g
expressed
cell
of
for
was determined of Shore et al.
histamine of
the
cells
ml
terminated
in
precipitates the method
0.5
PBS.
centrifuged
of
mast
in
with
mM 0.01%
90%.
release,
reaction
6.7
and
purity
than
suspended
the
CaCl2,
7.2,
The
more
histamine
incubated and
albumin.
was
cells/ml)
mM
pH
the to
for
effect
of
an 15 pH,
used.
The
mor-
mast invert-type
cells
were phase-
248
K. Sugiyama
& H. Furuta
2. Lactate dehydrogenase-release assay: Lactate dehydrogenase (LDH) activity was determined according to the fluorimetric method, as previously described by Roy et al. (13). The LDH release was assessed in relation to fluorescence intensity obtained after addition of 0.01% Triton X-100. 3. Chemicals: Dynorphin-(1-13) and leucine-enkephalin was obtained from the Protein Research Foundation (Osaka, Japan): disodium cromoglycate from Fujisawa Pharmaceutical Co. (Osaka, Japan); naloxone hydrochloride from Sankyo Company (Tokyo, Japan). Other chemicals of reagent grade were obtained from standard commercial sources.
(mean•}S.E.)
Fig.
2.
(B)
after
Phase-contrast treatment
micrographs with
5•~10-6
of M for
mast
cells
5 min. •~500.
The
(10-5
M)
release
of
2.
Results 1. Induction of histamine release by Dyn: Synthetic Dyn induced histamine release from isolated mast cells in a dose-dependent manner over the concentration range 10-710-5 M (Fig. 1). Phase contrast observations of mast cells responding to Dyn showed granule extrusion (Fig. 2). Lactate dehydrogenase did not release in the response to Dyn. Spontaneous release of histamine and LDH from mast cells was 4.2+0.8%. n=4
and
tively.
Fig.
1.
rat
Mast with
three
separate
cells
Dyn.
to
histamine
cells were
Values
while
the
release:
for
induced
by
incubated represent
Dyn
n=4.
relationships mast
respecby
n=4,
3.2•}0.4%, of
n=4, induced
70.2•}5.3, was
Dose-response
37•‹C
response
release
course
from
(1-13).
in
was LDH
Time
release
2.3•}0.5%,
histamine
for
The
histamine dynorphin10
the
mean•}S.E.
(A)
before
mm
of
experiments.
dynorphin-(1-13).
at
and
Histamine response
to
onset
Dyn
at
release
was
25•‹C,
min
(Fig. 3.
3). Effect
of
release
by
Dyn
(Fig. atures
4), the below
release
than
4.
Effect
7.5
and
(Fig.
pH's
added
addition
At temperhistamine
simple
omission
of
the
cells
ions release with 10-5
and
calcium
from
reduced this
various
was
of
EDTA
release times
Ca2+-free
the
release
histamine
at
the M,
M
addition
the
in
or
Ca2+-concen-
of
However,
Dyn
6.5
5•~10-4
medium
by
to of
was
than
increased from
at
shows
histarelease
Calcium
increase
influenced
Ca2+
Histamine
histamine
further
response.
7
the
calcium:
cell-suspending
(10-3 M). Figure
by
after
medium.
the
Fig.
4.
by with 37•‹C
3.
Time
course
of
dynorphin-(1-13). Dyn (•œ).
experiments.
histamine Mast
(5•~10-6
M)
Values
represent
for
release
cells 10 the
were
min
at
meari•}S.E.
of
induced
were
temperature by
preincubated
and
further
Dyn
(5•~10-6
at
various
incubation
represent
M) the
on
the
histamine
dynorphin-(1-13).
or
mean•}S.E.
cells
for
5 min,
temperatures
was (•œ)
Mast
made
for
without of
10
Dyn
three
min
(•›).
with Values
experiments.
When
induced
(•›) of
Effect by
incubated or
three
5.
induced
incubated 25•‹C
Effect
release
Fig. Fig.
exposure of reached a time, apbasal level
5).
maximum
not
a
CaCl2 was added after a 10-sec cells to Dyn, histamine release maximum and decayed with proaching asymptotically the
temperature
lower
effect of Ca2+ concentration
on
releasing
pH:
37•‹C. 40•‹C,
Dyn-induced
The of
The
the
at
249
by Dynorphin
peptide
Dyn-induced
7.3,
of
reaching
tration.
while
the
on
being above
at
the
declined
of and
for
was
enhanced (Fig. 6). increase
sec,
dependent
suppressed
higher
in
approached
addition
optimum
release
largely
10
temperature was
rapid histamine
release
after
Induced
suppressed.
pH
mine
within
optimum 15•‹C and
was
The
was
maximal
histamine
1
M)
and
reached
the
plateau
(5•~10-6
37•‹C
Release
(•œ)
or
with of
for
10
without
various three
of
pH
on
the
histamine
dynorphin-(1-13). min Dyn pH's.
experiments.
at (•›) Values
Mast 37•‹C in
with a
Dyn
suspending
represent
release cells
the
were
(5•~10-6 medium mean•}S.E.
M)
250
K. Sugiyama
& H. Furuta
obtained in the absence of calcium. 5. Effect of inhibitors: Cromoglycate inhibited histamine release stimulated by Dyn in a dose-dependent manner (Fig. 8a). This inhibition was dependent upon the incubation time with cromoglycate before addition of Dyn (Fig. 8b). The addition of cromoglycate 30 sec before Dyn inhibited
markedly histamine release from mast cells, but such inhibition was abolished after a 2-min incubation with cromoglycate. Naloxone (10-6-10-4 M) and leucineenkephalin (10-7-10-4 M) had little effect
Fig.
7.
Time
release Fig.
6.
Effect
induced
by
incubated in
a
of
at
on
the
histamine
dynorphin-(1-13). 37•‹C
medium
trations.
calcium
for
Mast
10
containing
Values
min
with
CaCl2
represent
the
cells
Dyn in
release
M)
concen-
mean•}S.E.
of
Fig.
8.
different
37•‹C
in
(•›).
CaCl2
periods
three
(•œ).
experiments.
was
of
calcium
decay
added
dynorphin-(1-13).
were
(5•~10-6
varying
course
by
Mast
a
Ca2+-free
time
Values
cells
medium
(5•~10-4
of
was
exposure
represent
the
the
of
incubated
Dyn added
to
histamine
stimulation
were with
M)
after
of after
at
(5•~10-6 at
M)
different
Dyn
(5•~10-6
mean•}S.E.
of
M) three
experiments.
Inhibition
added
to times
by mast after
cromoglycate cells exposure
30
of sec to
histamine
before cromoglycate.
addition
release of Values
induced
Dyn
by
(5•~10-6
represent
dynorphin-(1-13) M) .
the
b:
mean•}S.E.
Dyn
. (5•~10-6 of three
a: M)
Cromoglycate was
added
experiments
.
at
Histamine
Release
on the mast cells and on the Dyn-induced histamine release. Discussion We have shown here that Dyn induces histamine release from isolated rat peritoneal mast cells concomitantly with their degranulation. This histamine release is not accompanied by leakage of LDH. The response to Dyn is dependent on temperature and pH in the reaction medium. Calcium enhances the histamine release induced by Dyn. These data indicate that Dyn causes the release of histamine by an exocytotic mechanism. The histamine release induced by Dyn was augmented by the extracellular calcium, but a significant release of histamine persisted in the absence of calcium. Addition of EDTA in the absence of calcium did not abolish the histamine release by Dyn. However, we could not observe the restoration of histamine release by calcium after prolonged treatment of the cells with EDTA, as previously observed by Douglas and Ueda (14). The stimulant effect of Dyn would therefore be that it acts by utilizing extracellular calcium or by mobilizing cellular calcium which mediates the releasing response. Calcium-induced histamine release from the cells activated by Dyn in the Ca2+-free medium decayed with time and decreased on further exposure to Dyn. The time sequence of inactivation was similar to the process of desensitization to antigen described by Foreman and Garland (15). They suggested that mast cells limited the entry of calcium after stimulation by shutting-down Ca channels opened by the stimulus. A similar observation on inactivation of compound 48/ 80-treated cells has been shown by Cochrane et al. (16). However, the mechanism of Ca channel inactivation in mast cells is not yet understood. Cromoglycate has been shown to inhibit exocytosis from mast cells stimulated with a variety of agents such as antigen (17), compound 48/80 (18, 19), dextran (20) and neurotensin (21). The histamine release induced by Dyn was also inhibited by cromoglycate in a dose-dependent fashion. This inhibitory action was effective by the treatment with this agent for very short
Induced
by Dynorphin
251
periods of time. These conform to a familiar pattern observed with various other agents in mast cells (22). Cromoglycate might have a stabilizing effect on the mast cell membrane involved in exocytosis. However, the mechanism of action of cromoglycate in the inhibitory effect is at present uncertain. The actions of Dyn in bioassay systems such as guinea pig ileum and mouse was deferens are antagonized by naloxone (6), and it has been shown that Dyn acts as a κ-receptor
agonist(23).Dyn-induced
hista-
mine release was not inhibited by both naloxone and leucine-enkephalin. The histamine release from the mast cells was also not induced by leucine-enkephalin alone. These findings indicate that histamine release by Dyn is not probably mediated by opioid receptors of the mast cells. Certain basic biologically active peptides are known to acts as histamine releasers from mast cells (21, 24, 25). Dynorphin contains the leucine-enkephalin sequence at its Nterminus, followed by its C-terminus in which basic amino acids predominate. Dynorphin-(1-13) contains five basic amino acids in its peptide structure. Therefore, the releasing response to Dyn may reflect the stimulating effect of basic peptides observed by Johnson and Erdos (24). References 1 Goldstein, A., Fischli, W., Lowney, L.1., Hunkapiller, M. and Hood, L.: Pocine pituitary dynorphin: Complete amino acid sequence of the biologically active heptadecapeptide. Proc. Natl. Acad. Sci. U.S.A. 78, 7219-7223 (1981) 2 Tachibana, S., Araki, K., Ohya, S. and Yoshida, S.: Isolation and structure of dynorphin, an opioid peptide, from porcin duodenum. Nature 295, 339-340 (1982) 3 Goldstein, A. and Ghazarossian, V.E.: Immunoreactive dynorphin in pituitary and brain. Proc. Natl. Acad. Sci. U.S.A. 77, 6207-6210 (1980) 4 Hollt, V., Haarman, I., Bovermann, K., Jerlicz, M. and Herz, A.: Dynorphin-related immunoreactive peptides in rat brain and pituitary. Neurosci. Lett. 18, 149-153 (1980) 5 Watson, S.J., Akil, H., Gharzarossian, V.Z. and Goldstein, A.: Dynorphin immunocytochemical localization in brain and peripheral nervous system: Preliminary studies. Proc. Natl. Acad.
252
K. Sugiyama
Sci. U.S.A. 78, 1260-1263 (1981) 6 Goldstein, A., Tachibana, S., Lowney, L.I., Hunkapiller, M. and Hood, L.: Dynorphin- (1-13), an extraodinarilly potent opioid peptide. Proc. Natl. Acad. Sci. U.S.A. 76, 6666-6670 (1979) 7 Herman, B.H., Leslie, F. and Goldstein, A.: Behavioral effects and in vivo degradation of intraventriculary administered dynorphin- (1-13) and D-Ala2-dynorphin-(1-11) in rat. Life Sci. 27, 883-892 (1980) 8 Morley, J.E., Levine, A.S. and Silvis, S.E.: Endogenous opiates inhibit gastric acid secretion induced by central administration of thyrotropin releasing hormone (TRH). Life Sci, 29, 293-297 (1981) 9 Morley, J.E. and Levine, A.S.: Dynorphin- (113) induces spontaneous feeding in rats. Life Sci. 18, 1901-1903 (1981) 10 Sugiyama, K. and Furuta, H.: Histamine release induced by dynorphin-(1-13) from rat mast cells. Japan. J. Pharmacol. 33, Supp. 59P (1983) 11 Sugiyama, K.: Calcium-dependent histamine release with degranulation from isolated rat mast cells by adenosine 5'-triphosphate. Japan. J. Pharmacol. 21, 209-226 (1971) 12 Shore, P.A., Burkhalter, A. and Cohn, V.H., Jr.: A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther. 127, 182-186 (1959) 13 Roy, D., Moran, D.M., Bryant, V., Stevenson, R. and Stanworth, D.R.: Further studies on histamine release from rat mast cells in vitro induced by peptides. Biochem. J. 191, 233-237 (1980) 14 Douglas, W.W. and Ueda, Y.: Mast cell secretion (histamine release) induced by 48/80: Calcium dependent exocytosis inhibited strongly by cytochalasin only when glycolysis is ratelimiting. J. Physiol. (Lond.) 234, 97P-98P (1973) 15 Foreman, J.C. and Garland, L.G.: Desensitization in the process of histamine secretion induced by antigen and dextran. J. Physiol. (Lond.) 239, 381-391 (1974) 16 Cochrane, D.E., Distel, D.L., Lansman, J.B. and Paterson, B.M.: Stimulas-secretion coupling in
& H. Furuta
17
18
19
20
21
22
23
rat mast cells: Inactivation of extracellular calcium dependent secretion. J. Physiol. (Lond.) 323, 423-435 (1982) Garland, L.G.: Effect of cromoglycate on anaphylactic histamine release from rat peritoneal mast cells. Br. J. Pharmacol. 49, 128-130 (1973) Orr, T.S.C., Hall, D.E., Gwilliam, J.M. and Cox, J.S.G.: The effect of disodium cromoglycate on the release of histamine and degranulation of rat mast cells induced by compound 48/80. Life Sci. 10,805-809 (1971) Spataro, A.C. and Bosmann, H.B.: Mechanism of action of disodium cromoglycate on mast cells calcium ion influx after a histamine releasing stimulus. Biochem. Pharmacol. 25, 505-509 (1976) Garland, L.G. and Mongar, J.L.: Inhibition by cromoglycate of histamine release from rat peritoneal mast cells induced by mixtures of dextran, phosphatidylserine and calcium ions. Br. J. Pharmacol. 50, 137-143 (1974) Carraway, R., Cachrane, D.E., Lansman, J.B., Leeman, S.E., Paterson, B.M. and Welch, H.J.: Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels. J. Physiol. (Lond.) 323, 403414 (1982) White, J.R. and Pearce, L.: Effect of antiallergic compound an anaphylactic histamine secretion from rat peritoneal mast cells in the presence and absence of exogenous calcium. Immunology 46, 361-367 (1982) Oka, K., Negishi, K., Suda, M., Sawa, A., Fujino, M. and Wakimasu, M.: Evidence that dynorphin-(1-13) acts an agonist on opioid κ-receptors.
Eur.
J.
Pharmacol.
77,
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(1982) 24 Johnson, A.R. and Erdos, E.G.: Release of histamine from mast cells by vasoactive peptides. Proc. Soc. Exp. Biol. Med. 142, 1252-1256 (1973) 25 Theoharides, T.C. and Douglas, W.W.: Somatostatin induces histamine secretion from rat peritoneal mast cells. Endocrinology 102, 16371640 (1978)